Article Category: Reagents

10 Possible Causes of Weak Signals on Chemiluminescent Western Blot Images

Weak Signals on Chemiluminescent Western BlotsAre you seeing weaker than expected (hoped for. . .) signal on your chemiluminescent Western blot images with your digital imager? Not sure what could be causing this? Well, here is a list of 10 possible reasons why you might be seeing weak signals in chemiluminescent Western blot data:

  1. The chemiluminescent substrate does not have a fast enough rate of reaction.
  2. Not enough substrate was added to the blot.
  3. Membrane was placed on the detection system incorrectly.
  4. Blot was not detected or processed on the same day it was imaged.
  5. Blot was not kept uniformly wet during the entire image acquisition.
  6. Blot was exposed to film BEFORE imaging on a digital imager.
  7. Blot was imaged using incorrect sensitivity setting (learn about the easy-to-use Image Studio™ Software. Try FREE Image Studio Lite Western Blot Analysis Software to see just how easy it is!)
  8. Chemiluminescent substrate was too cold.
  9. Chemiluminescent substrate was not incubated for 5 minutes.
  10. Substrate was diluted.

Hum, that’s quite a list! For details on ways to eliminate or avoid these causes and get great results with your chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.

Use NEW! VRDye™ Secondary Antibodies to Correlate Near-Infrared Application Data with Microscopy and Flow Cytometry Data

VRDye Secondary Antibody IconsLI-COR is expanding its portfolio of reagents by offering VRDye™ 490, VRDye 549, and IRDye® 650 dye-labeled secondary antibodies and protein labeling kits. These new secondaries can be used for for a variety of applications, including immunofluorescence microscopy and flow cytometry. Just like our IRDye dye-labeled secondary antibodies, these new visible fluorescence antibodies are highly cross-adsorbed. The dyes are conjugated to the same antibodies as the existing IRDye secondary antibodies, which are used for Western blotting and In-Cell Western™ Assay applications. This gives researchers the ability to correlate microscopy and flow data with Western blot and cell-based assay data. The VRDye secondary antibodies are suitable for multiplex experiments when combined with other secondary antibodies labeled with proper fluorescent dyes and using instrumentation with appropriate excitation and detection capabilities.

Immunofluorescence staining of tubulin protein in HeLa cells.

Figure 1. Immunofluorescence staining of tubulin protein in HeLa cells. Cells were cultured on cover slips. After fixation and permeabilization, cells were incubated with rabbit anti-tubulin mAb (CST), followed by VRDye™ 490 Goat anti-Rabbit IgG (LI-COR P/N 926-49020). Nuclei were stained with DAPI. Image acquired with Olympus IX81 microscope.

Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides.

Figure 2. Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides. F98-EGFR tumors were snap-frozen in O.C.T. ™ compound and sectioned at 4-µm thickness. After fixation and permeabilization, cells were incubated with rabbit anti-EGFR mAb (CST), followed by detection with VRDye™ 549 Goat anti-Rabbit IgG (LI-COR P/N 926-54020). DAPI was used to stain the nuclei. Image acquired on Olympus IX81 microscope.

In addition, many researchers use labeled primary antibodies for flow cytometry. LI-COR now offers visible fluorescent dye protein labeling kits that are ideal for customers who need to label custom monoclonal antibodies for this application.

Visit our website for more information on these new visible fluorescence antibodies and protein labeling kits or to order them for your research.

Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

Example of Multiplex Western Blotting using the MPX Blotting SystemMultiplex Western blotting is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

  • Primary antibodies – to determine the primary antibody that has the right specificity and the right dilution for use
  • Antibody incubation times
  • Blocking conditions – which blocking buffer will give you the optimum results
  • Secondary antibodies – what dilutions is best to use without getting a lot of non-specific binding?
  • Or just about anything else you need to optimize!

Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

5 Technical Tips for Chemiluminescent Western Blotting Success with the C-DiGit® Scanner

In this short video, Jessica talks about 5 tips to help ensure that imaging chemiluminescent Western blots on the C-DiGit Blot Scanner is a success – the first time and always!

Here’s a recap of the tips of those five technical tips:

  1. Install Image Studio™ Software on your computer before connecting the C-DiGit Blot Scanner.
  2. Incubate using room temperature substrate.
  3. Wrap your blot so it stays wet during the scan.
    1. Remember to place your blot protein side down.
    2. If sensitivity is an issue, use WesternSure® PREMIUM or SuperSignal® West Femto Chemiluminescent Substrate.
  4. Start with high sensitivity scan for your first scan and then work from there.
  5. Image on the C-DiGit Scanner first and then exposure your film.

C-DiGit Blot ScannerHappy Blotting!

Rethinking the Traditional Western Blot

Traditional Western blotting is a labor-intensive process that includes gel electrophoresis, protein transfer to a blotting membrane, incubation with primary and secondary antibodies, and chemiluminescent or fluorescent detection of target proteins. (View a typical Western blotting workflow.) Day-to-day reproducibility is poor, because small variations in lysate preparation, gel loading, electrophoresis, transfer, and detection are unavoidable sources of technical variability.

Snapshot of In-Cell Western Assay MethodThe In-Cell Western™ (ICW) Assay, a quantitative immunofluorescent method, is an alternative to traditional Western blots that increases both reproducibility and sample throughput. (View a typical ICW workflow.)

We recently hosted a webinar called “Rethinking the Traditional Western Blot”, during which John Lyssand, PhD, from LI-COR Biosciences, discussed the In-Cell Western Assay and an example of its use in neuroscience research, in this case, Alzheimer’s Disease. The In-Cell Western Assay enables screening and analysis of many more samples in each experiment, eliminates error-prone protocol steps, and delivers higher reproducibility for biological and technical replicates.

ICW Use: Tau Protein Accumulation and InhibitionThe data presented demonstrated how ICW assays were used in Alzheimer’s Disease research to screen HSP90 inhibitors for their effectiveness in reducing tau activity levels. Dr Lyssand discussed how and why the In-Cell Western Assay is superior to traditional methods for screening of cell samples.

If you didn’t have a chance to join us in September for “Rethinking the Traditional Western blot”, you can view this webinar online and on-demand. Check out the information on In-Cell Western assays on our website. You can also read Professor Dickey’s white paper as cited above that outlines how he and his group used higher throughput method to study Alzheimer’s Disease.

New Cell Stain Increases Ease of Use for In-Cell Western™ Normalization

CellTag 700 Stain ICW Kits for Quantitative Cell Signaling AnalysisHave you ever wanted to try an in-cell ELISA but you just weren’t sure how to get started? With the new LI-COR® CellTag™ 700 Stain, a near-infrared fluorescent, non-specific cell stain that provides accurate normalization to cell number, you have a easier — and more affordable — way to try this powerful application. CellTag 700 Stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers (see Figure 1 below). CellTag 700 Stain is applied to the cells during incubation with IRDye® 800CW secondary antibody, and enables accurate measurement of target protein levels with much higher throughput than Western blotting.

CellTag 700 Stain - Linear Relationship between Fluorescence and Cell Number.

Figure 1. Linear Relationship between Fluorescence and Cell Number. Two-fold serial dilutions of A431 and NIH/3T3 cells were plated in 96-well plate, then fixed, permeabilized, stained with CellTag 700 Stain, and detected with Odyssey Classic (Resolution: 169um; Quality: medium; Focus offset: 4.0mm; Intensity: 5). The Trim Signals were used to generate the graphs.

CellTag 700 Stain ICW Kits offer a convenient way to try cell-based In-Cell Western Assays. Each kit includes blocking buffer, IRDye® 800CW secondary antibody for detection of a specific protein target in the 800 nm channel, and CellTag 700 Stain to normalize well-to-well variations in cell number. This cost-effective normalization method makes quantification of the target protein more precise.
In-Cell Western Normalization with CellTag 700 Stain in EGF-stimulated A431 Cells.Figure 2. In-Cell Western Assay with CellTag 700 Stain in EGF-stimulated A431 Cells. (Go to the CellTag 700 Stain Overview page for more details on this data).

Try one of our new In-Cell Western Assay Kits with CellTag 700 Stain today and find out just how easy it is to perform fast, cost-effective cell-based Western assays.

Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.


Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.


Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.


Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.


Check out some of our Publications Lists for:

Analyze Glycoproteins with Sensitive, Quantitative Infrared Fluorescent Techniques

O-Linked Glycan StructureGlycosylation is one of the most common and important events in post-translational modification. Over half of all proteins are believed to be glycosylated, and the resulting glycoconjugates play an important role in many biological processes. They have been connected to instances of cancer development, retrovirus infection, and other diseases. In an effort to understand these diseases, glycoprotein analysis has become a growing area of research. (See examples of typical glycan structures.)

Analysis of glycoproteins requires sensitive and quantitative applications. LI-COR offers a single, optimized solution using the Odyssey® Systems and IRDye® labeled conjugates to analyze glycoproteins. This solution provides sensitive and quantitative results using two-color near-infrared detection at 700 nm and 800 nm wavelengths. Operating at this wavelength produces lower background from biological materials, buffer components, and standard membranes used in Western blotting and lectin binding applications and, thus, superior data.

Outlined below are a variety of applications for several one-color, visible glycoprotein applications that have been adapted to near-infrared fluorescence detection on an Odyssey Imaging System:

Read Glycoprotein Detection with the Odyssey Infrared Imaging System for more indepth information on using your Odyssey Infrared Imaging System for glycobiology research.

Click Chemistry Reagents from LI-COR® for Biomolecule Labeling

Biomolecule labeling continues to be a cornerstone feature of many in vitro and in vivo biological experiments. Click Chemistry has recently emerged as a convenient, versatile, and reliable method for labeling a wide variety of molecules for applications ranging from biomarker isolation to assay development.
Click Chemistry Workflow
LI-COR now offers a portfolio of Click Chemistry reagents for copper-catalyzed and copper-free methods. These products offer researchers flexibility to choose the correct reagent for a diverse array of applications. LI-COR Click Chemistry reagents include IRDye® 800CW, IRDye 680RD, and IRDye 650 infrared fluorescent dyes labeled with DBCO, azide, or alkyne groups.

Click Chemistry utilizes pairs of reagents that exclusively react with each other and are effectively inert to naturally-occurring functional groups such as amines. Unlike affinity interactions such as streptavidin-biotin, Click Chemistry forges covalent bonds between the reacting partners to deliver stable bioconjugates.

Click Chemistry reactions can be categorized into two separate groups, copper-catalyzed or copper-free. Copper-catalyzed Click Chemistry is used for initiating reactions between azides and alkynes. These reactions are also known as Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC). Although they initiate and accelerate Click Reactions, copper catalysts are cytotoxic and inappropriate for use in living systems.

Watch this informative webinar on IRDye Infrared Dye Reagents for Click Chemistry.

Click Chemistry Reagents Labeled with DBCO Groups Allow for Copper-Free Biomolecule Labeling Reactions

LI-COR now offers Click Chemistry reagents for copper-catalyzed and copper-free methods. One group of products within this portfolio includes IRDye® infrared dyes labeled with DBCO groups, which can be used for copper-free methods.

The dibenzocyclooctyne group (DBCO) allows copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. DBCO groups will preferentially and spontaneously label molecules containing azide groups (—N3). Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. Reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (—SH, thiol).
Click Chemistry Copper-Free Reaction

Click chemistry reagents with DBCO groups are available in 0.5 mg and 5 mg sizes for 3 dyes: IRDye 80CW, IRDye 680RD, and IRDye 650. IRDye 800CW DBCO also comes in a 50-mg pack size. For other sizes, contact LI-COR Custom Services.

Watch this 18-minute webinar to learn more about Click Chemistry applications and the new LI-COR® Click Chemistry reagents.